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WO2022174065A1 - Méthodes pour induire une réponse à l'interféron par régulation de morc3 - Google Patents

Méthodes pour induire une réponse à l'interféron par régulation de morc3 Download PDF

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WO2022174065A1
WO2022174065A1 PCT/US2022/016162 US2022016162W WO2022174065A1 WO 2022174065 A1 WO2022174065 A1 WO 2022174065A1 US 2022016162 W US2022016162 W US 2022016162W WO 2022174065 A1 WO2022174065 A1 WO 2022174065A1
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morc3
interferon
seq
ifn
disorder
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WO2022174065A8 (fr
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Russell Eaton VANCE
Moritz MATTHIAS GAIDT
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Priority to US18/263,760 priority patent/US20240076666A1/en
Publication of WO2022174065A1 publication Critical patent/WO2022174065A1/fr
Publication of WO2022174065A8 publication Critical patent/WO2022174065A8/fr
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function

Definitions

  • Type I interferons are cytokines that have potent immunomodulatory effects.
  • IFNs induce anti-viral responses, suppress autoimmune inflammation, and activate cytotoxic lymphocytes.
  • the innate immune system detects pathogens by employing germ-line-encoded pattern-recognition-receptors (PRRs) that sense pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide and nucleic acids.
  • PRRs germ-line-encoded pattern-recognition-receptors
  • PAMPs pathogen-associated molecular patterns
  • HSV-1 Herpes Simplex Virus-1
  • STING cyclic-GMP-AMP synthase
  • TNK1 TANK Binding Kinase 1
  • IKKe I-kappa-B kinase e
  • IRF3/7 interferon regulatory factors 3 and 7
  • IFNs bind to the IFN alpha and beta receptor (IFNAR) to induce transcription of anti-viral interferon-stimulated genes (ISGs).
  • the present invention is directed to a method of increasing or decreasing the amount of endogenous interferon in a subject, which comprises administering to the subject a MORC3 therapeutic agent or modulating the activity of MRE in the subject.
  • the MORC3 therapeutic agent is an siRNA, an ATPase inhibitor (e.g ., a small molecule ATPase inhibitor), a MORC3 protein, or an ICPO protein.
  • the MORC3 therapeutic agent is an siRNA, an ATPase inhibitor (e.g., a small molecule ATPase inhibitor), a MORC3 protein having at least about 90% sequence identity to SEQ ID NO: 1, or an ICPO protein having at least about 95% sequence identity to SEQ ID NO: 2.
  • the MORC3 therapeutic agent is an siRNA, an ATPase inhibitor (e.g, a small molecule ATPase inhibitor), a MORC3 protein, or an ICPO protein.
  • the MORC3 therapeutic agent is a MORC3 inhibitor.
  • the MORC3 therapeutic agent is a MORC3 activator.
  • the MORC3 therapeutic agent stabilizes expression of MORC3.
  • the MORC3 protein comprises a sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:
  • the ICPO protein comprises a sequence that has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 2.
  • the sequence of the MRE in the subject at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:
  • the subject is in need thereof.
  • the present invention is directed to a method of treating an IFN Excess Disorder in a subject, which comprises administering to the subject a MORC3 therapeutic agent.
  • the interferon disorder is an IFNBl Disorder.
  • the present invention is directed to a method of treating an IFN Excess Disorder in a subject, which comprises increasing the amount of MORC3 in the subject.
  • the subject is in need thereof.
  • the IFN Excess Disorder is an autoimmune or inflammatory disease.
  • the IFN Excess Disorder is rheumatoid arthritis, psoriasis, vitiligo, hypothyroidism, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, myasthenia gravis, Addison disease, celiac disease, polymyositis, or superimposed autoimmune hepatitis.
  • the autoimmune or inflammatory disease is multiple sclerosis.
  • the multiple sclerosis is primary progressive multiple sclerosis or relapsing-remitting multiple sclerosis.
  • the IFN Excess Disorder is an IFNBl Disorder.
  • the MORC3 therapeutic agent is a MORC3 protein. In some embodiments, the MORC3 therapeutic agent is a MORC3 activator. In some embodiments, the MORC3 therapeutic agent stabilizes expression of MORC3. In some embodiments, the MORC3 protein comprises a sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the method further comprises inhibiting or reducing the activity of MRE in the subject.
  • sequence of the MRE in the subject at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 3.
  • the present invention is directed to a method of treating IFN Deficiency Disorder in a subject, which comprises administering to the subject a MORC3 therapeutic agent. In some embodiments, the present invention is directed to a method of treating IFN Deficiency Disorder in a subject, which comprises decreasing the amount of MORC3 in the subject.
  • the IFN Deficiency Disorder is a viral infection. In some embodiments, the viral infection is caused by a herpes virus, a hepatitis virus, or a coronavirus. In some embodiments, the IFN Deficiency Disorder is a cancer.
  • the cancer is a leukemia, a lymphoma, a melanoma, a sarcoma, or an adenocarcinoma.
  • the subject is in need thereof.
  • the IFN Deficiency Disorder is a viral infection such as that caused by a herpes virus, a hepatitis virus, or a coronavirus.
  • the IFN Deficiency Disorder is a cancer such as a leukemia, a lymphoma, a melanoma, a sarcoma, or an adenocarcinoma.
  • the cancer is colon cancer.
  • the IFN Deficiency Disorder is an IFNBl Disorder.
  • the MORC3 therapeutic agent is a MORC3 inhibitor such as an siRNA, an ATPase inhibitor (e.g ., a small molecule ATPase inhibitor), or an ICP0 protein.
  • the MORC3 therapeutic agent is a MORC3 inhibitor such as an siRNA, an ATPase inhibitor (e.g., a small molecule ATPase inhibitor), or an ICP0 protein.
  • the ICP0 protein comprises a sequence that has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 2.
  • the present invention is directed to a method of treating an interferon disorder (alternatively referred to herein as a “MORC3 -modulated disease” as it is modulated by MORC3) in a subject, which comprises administering to the subject a MORC3 therapeutic agent.
  • the MORC3 therapeutic agent is an siRNA, an ATPase inhibitor (e.g, a small molecule ATPase inhibitor), a MORC3 protein, or an ICPO protein.
  • the method comprises decreasing the amount of endogenous interferon in the subject by administering the MORC3 therapeutic agent, wherein the MORC3 therapeutic agent is a MORC3 activator.
  • the method comprises decreasing the amount of endogenous interferon in the subject by administering the MORC3 therapeutic agent, which is a MORC3 protein, and/or stabilizes the expression of MORC3. In some embodiments, the method comprises increasing the amount of endogenous interferon in the subject by administering the MORC3 therapeutic agent, wherein the MORC3 therapeutic agent is a MORC3 inhibitor. In some embodiments, the method comprises increasing the amount of endogenous interferon in the subject by administering the MORC3 therapeutic agent, which is an siRNA, an ATPase inhibitor (e.g, a small molecule ATPase inhibitor), and/or an ICPO protein.
  • the MORC3 therapeutic agent which is an siRNA, an ATPase inhibitor (e.g, a small molecule ATPase inhibitor), and/or an ICPO protein.
  • the MORC3 protein comprises a sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:
  • the ICPO protein comprises a sequence that has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 2.
  • the subject is in need thereof.
  • the present invention is directed to a method of treating an interferon disorder (alternatively referred to herein as a “MORC3 -modulated disease” as it is modulated by MORC3) in a subject, which comprises administering to the subject a MORC3 therapeutic agent.
  • the MORC3 -modulated disease is an autoimmune or inflammatory disease.
  • the autoimmune or inflammatory disease is multiple sclerosis.
  • the multiple sclerosis is primary progressive multiple sclerosis or relapsing-remitting multiple sclerosis.
  • the MORC3 -modulated disease is rheumatoid arthritis, psoriasis, vitiligo, hypothyroidism, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, myasthenia gravis, Addison disease, celiac disease, polymyositis, or superimposed autoimmune hepatitis.
  • the MORC3 therapeutic agent is a MORC3 protein.
  • the MORC3 protein comprises a sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about
  • the MORC3 therapeutic agent is a MORC3 activator and/or stabilizes the expression of MORC3.
  • the subject is in need thereof.
  • the present invention is directed to a method of treating an interferon disorder (alternatively referred to herein as a “MORC3 -modulated disease” as it is modulated by MORC3) in a subject, which comprises administering to the subject a MORC3 therapeutic agent.
  • the MORC3 -modulated disease is cancer.
  • the cancer is a leukemia, a lymphoma, a melanoma, a sarcoma, or an adenocarcinoma.
  • the cancer is colon cancer.
  • the MORC3 -modulated disease is a viral infection.
  • the viral infection is caused by a herpes virus, a hepatitis virus, or a coronavirus.
  • the MORC3 therapeutic agent is a MORC3 inhibitor such as an siRNA, an ATPase inhibitor (e.g, a small molecule ATPase inhibitor), or an ICP0 protein.
  • the ICP0 protein comprises a sequence that has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 2.
  • the subject is in need thereof.
  • the present invention is directed to an assay method for determining whether a candidate compound is a MORC3 inhibitor, which comprises contacting the candidate compound with a monocyte and measuring any interferon response induced thereby in the monocyte.
  • the assay method further comprises contacting the candidate compound with a genetically modified monocyte that deficient in MORC3 activity, measuring any interferon response induced thereby in the genetically modified monocyte, and comparing the interferon response in the monocyte to the interferon response in the genetically modified cell.
  • the present invention is directed to a method of increasing interferon expression by a cell, which comprises (a) the decreasing amount of MORC3 in the cell, (b) increasing MRE activity in the cell, or both (a) and (b).
  • the MORC3 is decreased by administering an ICP0 protein to the cell or contacting the MORC3 with an ATPase inhibitor (e.g, a small molecule ATPase inhibitor), or inactivating the MORC3 gene in the cell using recombinant techniques, or silencing the MORC3 gene with an siRNA.
  • the ICP0 protein comprises a sequence that has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 2.
  • a method of decreasing interferon expression by a cell which comprises (a) increasing the amount of a MORC3 protein in the cell, (b) reducing MRE activity in the cell, or (c) both (a) and (b).
  • the amount of MORC3 is increased by administering a MORC3 protein to the cell and/or increasing the expression of a MORC3 protein in the cell using recombinant techniques.
  • the MORC3 protein comprises a sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 1.
  • the MRE activity is reduced by recombinant techniques, e.g ., modifying or deleting the MRE sequence in the cell.
  • the present invention provides a MORC3 protein for use as a medicament. In some embodiments, the present invention provides a MORC3 protein for use in the treatment of an interferon disorder in a subject. In some embodiments, the interferon disorder is an IFN Deficiency Disorder. In some embodiments, the IFN Deficiency Disorder is a viral infection. In some embodiments, the viral infection is caused by a herpes virus, a hepatitis virus, or a coronavirus. In some embodiments, the IFN Deficiency Disorder is a cancer.
  • the cancer is a leukemia, a lymphoma, a melanoma, a sarcoma, or an adenocarcinoma.
  • the IFN Deficiency Disorder is a viral infection such as that caused by a herpes virus, a hepatitis virus, or a coronavirus.
  • the IFN Deficiency Disorder is a cancer such as a leukemia, a lymphoma, a melanoma, a sarcoma, or an adenocarcinoma.
  • the cancer is colon cancer.
  • the MORC3 protein comprises a sequence that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 1.
  • the present invention provides an ICP0 protein for use as a medicament. In some embodiments, the present invention provides an ICP0 protein for use in the treatment of an interferon disorder in a subject. In some embodiments, the interferon disorder is an IFN Excess Disorder. In some embodiments, the IFN Excess Disorder is rheumatoid arthritis, psoriasis, vitiligo, hypothyroidism, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, myasthenia gravis,
  • the autoimmune or inflammatory disease is multiple sclerosis.
  • the multiple sclerosis is primary progressive multiple sclerosis or relapsing-remitting multiple sclerosis.
  • the ICPO protein comprises a sequence that has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 2.
  • FIG. 1-a - FIG. 1-f Virulence factor-triggered immune sensing. (FIG. 1-a, FIG.
  • BLaERl monocytes expressing doxycycline-inducible virulence factors were stimulated with doxycycline for 24 h.
  • Gene-expression as quantified by q-RT-PCR is depicted as mean ⁇ SEM of three independent experiments (FIG. 1-d).
  • Transcriptomic changes were profiled by RNA-seq in three independent experiments and were analyzed by principal component analysis (PC A) of variance stabilizing transformed counts (FIG. 1-e) and heatmap of log normalized counts of the top 40 most variable genes (column normalized) (FIG. 1-f).
  • FIG. 2-a - FIG. 2-g MORC3 is a novel negative regulator of IFN.
  • FIG. 2-b Schematic of genome wide CRISPR screen to identify negative regulator of IFN.
  • FIG.2-d IFNAR1 –/– IFNAR2 –/– STING –/– SP100 –/– BLaER1 monocytes (lacking factors that would otherwise restrict ⁇ ICP0 mutant virus) were infected with HSV-1 for 3 h.
  • FIG.2-f, FIG.2-g Transcriptional changes in BLaER1 monocytes as detected by RNA-seq in three independent experiments are depicted by PCA analysis (FIG.2-f) and heatmap (FIG.2-g). These data are partially duplicated from FIG.1-e.
  • FIG.3-a ⁇ FIG.3-h Dual functions of MORC3 allows self-guarding.
  • FIG.3-b BLaER1 monocytes were infected with HSV-1 and viral progeny were quantified after 48 h.
  • FIG.3-c Two proposed functions of MORC3 that allow self- guarding.
  • FIG.3-d, FIG.3-e Transcriptomic changes in BLaER1 monocytes as detected by RNA-seq in three independent experiments: heatmap of log normalized counts of the top 40 most variable genes (column normalized) (FIG.3-d) or PCA of variance stabilizing transformed counts (FIG.3-e). Data in (FIG.3-e) is partially duplicated from FIG.2-f.
  • FIG.3-f Log transcripts per million (TPM) of genes in BLaER1 monocytes of genes clustered near IFNB1 on chromosome 9 as detected by RNA-seq. Depicted are means of three independent experiments. All protein coding genes within this region are depicted.
  • FIG.4-a ⁇ FIG.4-g Positional repression of IFNB1 explains IFN de-repression in MORC3 deficient cells.
  • FIG.4-a BLaER1 monocytes of the indicated genotypes were transfected with DNA for 12 h. Gene-expression as measured by q-RT-PCR of one representative clone of two per genotype is depicted as mean ⁇ SEM of four independent experiments.
  • FIG. 4-b Log transcripts per million (TPM) of IFN genes in BLaERl monocytes were detected by RNAseq in three independent experiments.
  • TPM Log transcripts per million
  • FIG. 4-g Overview of virulence-factor-triggered immune signaling.
  • FIG. 5-a - FIG. 5-c Redundancy between DNA- and ICPO-mediated sensing of HSV-1 in BLaERl monocytes.
  • BLaERl monocytes were infected with HSV-1 for 24 h or transfected with DNA for 3 h.
  • Gene-expression analysis or viral titer in supernatant of one representative clone of two per genotype (except WT) is depicted as mean ⁇ SEM of three independent experiments.
  • * p ⁇ 0.05; ** p ⁇ 0.01; ns not significantly different than WT, tested by two-way ANOVA and Dunnett’s post hoc test.
  • LOD limit of detection.
  • FIG. 6-a - FIG. 6-b Redundancy between DNA- and ICPO-mediated sensing of HSV-1 in THP1 and U937 cells.
  • PMA-differentiated THP1 or U937 human myeloid- like cells were infected with HSV-1 for 24 h or transfected with 2'-3'-cGAMP for 4 h.
  • Gene-expression analysis or viral titer in supernatant is depicted as mean ⁇ SEM of three independent experiments.
  • ND not detected.
  • * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001; ns not significantly different than WT, tested by two-way ANOVA and Dunnett’s post hoc test.
  • FIG. 7-a - FIG. 7-c Virulence factors induce IFN in monocytes independently of PRR-signaling hubs.
  • BLaERl monocytes, PMA-differentiated THP1 or U937 cells expressing doxycycline-inducible virulence factors were stimulated with doxycycline for 24 h.
  • Gene-expression as quantified by q-RT-PCR is depicted as mean ⁇ SEM of three independent experiments.
  • IFNB1 expression levels in FIG. 7-a are partially duplicated from FIG. 1-d.
  • FIG. 8-a - FIG. 8-d Virulence factor activity is required for IFN induction.
  • FIG. 8-c Transcriptomic changes in BLaERl monocytes as detected by RNA-seq were analyzed by PCA of variance stabilizing transformed counts.
  • FIG. 8-d Modules from the Molecular Signatures Database that were found to be enriched in differentially expressed genes upon virulence factor expression in BLaERl monocytes. Background gene set includes all genes that have base mean expression of at least 1 in the virulence factor expressing condition.
  • FIG. 9-a - FIG. 9-h Validation of MORC3 as a repressor of IFN.
  • FIG. 9-a Cell sorting strategy for the genome-wide CRISPR screen to identify negative regulators of IFN.
  • FIG. 9-b U20S cells were infected with HSV-1 for 24 h. One representative immunoblot of two is shown.
  • FIG. 9-g Immunoblot of BLaERl monocytes of indicated genotypes.
  • FIG. 9-h Modules from the Molecular Signatures Database that were found to be enriched in differentially expressed genes in MORC3 -/- BLaERl monocytes. Background gene set includes all genes that have base mean expression of at least 1 in MORC3 -/- BLaERl monocytes.
  • FIG. 10-a - FIG. 10-f Characterization of MORC3 deficiency in THP1, U937 and HCT116 cells.
  • FIG. 11-a — FIG. 11-b MORC3 deficiency leads to de-repression of a gene cluster at chromosome 9.
  • FIG. 11-a Adjusted p-values of differentially expressed genes between MORC3 -/- IFNARl -/- IFNAR2 -/- and IFNARl -/- IFNAR2 -/- mCherry expressing cells are depicted. Data point sizes represent normalized effect size, calculated as the effect size multiplied by the normalized mean counts of all MORC3 –/– IFNAR1 –/– IFNAR2 –/– and IFNAR1 –/– IFNAR2 –/– expressing cells.
  • FIG.11-b BLaER1 monocytes were stimulated with indicated PAMPs for 3 h or 24 h. Gene-expression analysis is depicted as mean ⁇ SEM of five independent experiments.
  • FIG.12-a Quantification of expression of ERV families. ERV family expression was quantified with RepEnrich2 in IFNAR1 –/– IFNAR2 –/– mCherry vs MORC3 –/– IFNAR1 – /– IFNAR2 –/– monocytes. ERVs with an FDR ⁇ 0.05 are highlighted in red. The recommended RepEnrich2-EdgeR pipeline did not detect any regulation of ERVs upon MORC3 deficiency.
  • FIG.13-a ⁇ FIG.13-f Positional de-repression of IFNB1 explains IFN induction by virulence factors.
  • FIG.13-a BLaER1 monocytes were transfected with DNA for 12 h. Cytokine secretion from one representative clone of two per genotype is depicted as mean ⁇ SEM of three independent experiments.
  • FIG.13-b Mean log transcripts per million (TPM) of a gene cluster at chromosome 9 in BLaER1 monocytes . Mean TPM is calculated for each condition across three experiments. All protein coding genes within this region are depicted.
  • FIG.13-c PCA of variance stabilizing transformed gene counts in BLaER1 monocytes in three independent experiments. Data is partially duplicated from FIG.3-e.
  • FIG.13-d Log transcripts per million (TPM) of IFN genes in BLaER1 monocytes upon virulence factor expression were detected by RNAseq in three independent experiments.
  • FIG.13-f BLaER1 monocytes expressing doxycycline-inducible virulence factors were stimulated with doxycycline for 24 h. Transcriptomic changes as detected by RNA-seq in three independent experiments were analyzed PCA. Data is partially duplicated from FIG.1-f. [0038] FIG.14-a ⁇ FIG.14-j: A MORC3-regulated DNA element explains positional IFNB1 activation.
  • FIG.14-a Adjusted p-values of increased accessibility in MORC3 –/– IFNAR1 –/– IFNAR2 –/– vs IFNAR1 –/– IFNAR2 –/– BLaER1 monocytes of non-promoter- associated ATAC-seq peaks from three independent experiments. Peaks are labeled according to the adjacent gene and peaks overlapping with the MRE are highlighted in red.
  • FIG.14-b A MORC3-regulated DNA element located in an intron of FOCAD. Sum of normalized ATAC-seq reads from three independent experiments is depicted. Sequencing overlapping a MRE ⁇ allele is indicated.
  • FIG.14-c STAT1 –/– STAT2 –/– Cas9 (“WT”) or STAT1 –/– STAT2 –/– MRE ⁇ / ⁇ Cas9 BLaER1 monocytes of indicated genotype were stimulated with PAMPs for 3 h or left untreated. Gene expression is shown as mean + SEM of three independent experiments.
  • FIG.14-d Schematic overview of tracking allele-specific gene expression.
  • FIG.14-e Allele-specific gene expression STAT1 –/– STAT2 –/– Cas9 or STAT1 –/– STAT2 –/– MRE +/ ⁇ Cas9 cells that were transduced with indicated sgRNAs and stimulated with DNA for 3 h if indicated. Data is depicted as mean + SEM of three independent clones (harboring different IFNB1 indels) as average from three experiments.
  • FIG.14-f Transcriptional changes in BLaER1 monocytes as detected by RNA-seq in two independent experiments are depicted by heatmap (column normalized).
  • High-confidence MORC3 targets (genes de-repressed in MORC3 –/– IFNAR1 –/– IFNAR2 –/– , MORC3 –/– IFNB1 –/– and MORC3 –/– STAT1 –/– STAT2 –/– Cas9) cells are depicted.
  • FIG.14-g, FIG.14-h STAT1 –/– STAT2 –/– Cas9 (“WT”) or STAT1 –/– STAT2 –/– MRE ⁇ / ⁇ Cas9 BLaER1 monocytes expressing doxycycline-inducible virulence factors were stimulated with doxycycline for 24 h.
  • FIG.14-i STAT1 –/– STAT2 –/– Cas9 (“WT”) or STAT1 –/– STAT2 –/– MRE ⁇ / ⁇ Cas9 BLaER1 were infected with HSV-1 at indicated MOI for 4-5 h. Gene expression is shown as mean + SEM of five independent experiments.
  • FIG.15-a ⁇ FIG.15-h Validation of the MORC3-regulated DNA element.
  • FIG. 15-a Protein expression of indicated BLaER1 monocytes was analyzed by immunoblot.
  • FIG.15-b Consensus sequences of amplicon sequencing at the MRE locus from STAT1 –/– STAT2 –/– MRE ⁇ / ⁇ BLaER1 Cas9 cells were aligned to the WT reference. 3166bp were omitted from the reference sequence.
  • FIG.15-c Protein expression of indicated BLaER1 monocytes was analyzed by immunoblot.
  • FIG.15-d Gene expression of indicated BLaER1 monocytes is depicted as mean ⁇ SEM of three independent experiments from one representative clone of two (MORC3 –/– MLLT3 –/– IFNAR1 –/– IFNAR2 –/– and MORC3 –/– FOCAD –/– IFNAR1 –/– IFNAR2 –/– ) or one (IFNAR1 –/– IFNAR2 –/– and MORC3 –/– IFNAR1 –/– IFNAR2 –/– ) per genotype.
  • ns not significantly different than the IFNAR1 –/– IFNAR2 –/– condition, tested by two-way ANOVA and Dunnett’s post hoc test. From top to bottom, the sequences are SEQ ID NO: 59 + SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62.
  • FIG.15-e Protein expression of indicated BLaER1 monocytes was analyzed by immunoblot.
  • FIG.15-g STAT1 –/– STAT2 –/– Cas9 (“WT”) or STAT1 –/– STAT2 –/– MRE ⁇ / ⁇ BLaER1 Cas9 were infected with HSV-1 at indicated MOI for 4-5 h. One representative immunoblot of two is depicted.
  • FIG.15-h Peripheral blood human monocytes were nucleofected with indicated sgRNA:Cas9 complexes and differentiated with M-CSF for 5 days. Gene expression from three independent donors is depicted as mean ⁇ SEM.
  • FIG.16-a FIG.16-c: Targeting of Morc3 in vivo results in interferon induction and tumor clearance.
  • FIG.16-a Mice harboring a floxed Morc3 gene were crossed to mice harboring a tamoxifen-inducible Cre (CreERT2). These mice were fed chow containing tamoxifen for 10 days.
  • FIG.16-c The mice described in FIG.16-a were implanted with 500,000 MC38-Luc tumor cells, as in FIG.16-b, but instead of oral provision of tamoxifen in the chow, tamoxifen was injected intraperitoneally once per day on days 1-5. Mice harboring CreERT2 (green symbols) that delete Morc3 exhibit enhanced tumor control whereas control (CreERT2-negative) mice (red symbols) that retain Morc3 expression exhibit normal tumor growth.
  • MORC3 MORC family CW-type zinc finger protein 3
  • HSV-1 Herpes Simplex Virus-1
  • YP_009137074.1 results in robust induction of anti-viral type I interferon (IFN).
  • IFN anti-viral type I interferon
  • a CRISPR-screen identified the ICP0 target MORC3 as an important negative regulator of IFN. Loss of MORC3 recapitulates the IRF3/7-independent IFN response induced by ICP0.
  • ICP0 degrades MORC3, which leads to de- repression of the IFNB1 locus in a genomic location-specific manner to drive the anti- viral IFN response.
  • MORC3 is also a direct restriction factor of HSV-1.
  • IFN induction by HSV-1 can occur independently of canonical PRRs.
  • IFN induction in response to transfected dsDNA depended entirely on the STING pathway and downstream TBK1/IKK ⁇ and IRF3/7 signaling components (FIG.1-a, FIG.5-a).
  • PRR-independent innate immune pathway detects HSV-1 infection in human monocytes.
  • the possibility that the immune system is activated by a viral virulence factor was considered.
  • a key virulence factor of HSV-1 is the SUMO-directed E3 ubiquitin ligase ICP0, which facilitates lytic replication by recognizing sumoylated ND10 nuclear body proteins, targeting them for proteasomal degradation.
  • ICP0 was necessary for induction of PRR-independent IFN in BLaER1 monocytes (FIG.1-b, FIG.5-b) while loss of ICP0 only modestly affected HSV-1 replication (FIG.5-c). Induction of IFN by HSV-1 was abrogated only by deleting both the STING pathway from host cells and ICP0 from HSV-1 (FIG.1-b). Thus, during HSV-1 infection of BLaERl monocytes, two redundant pathways induce IFN: STING- mediated viral DNA-sensing and another innate sensing pathway, which requires ICP0 (FIG. 1-c). This redundancy was confirmed in THP1 and U937 cells, two other human monocyte-like cell culture models. As in BLaERl monocytes, IFN was induced by either host STING or viral ICP0 and was abrogated only in absence of both (FIG. 6-a, FIG. 6-b).
  • ICP0 was ectopically expressed in human monocytes utilizing a doxycycline-inducible lentiviral system. Mirroring HSV-1 infection, ICP0 expression led to an IFN response that was independent of the PRR-signaling hubs TBKI/IKKe, IRF3/7 and IKKa/b (FIG. 1-d, FIG. 7-a). Thus, ICP0 is not only important but is also sufficient for IFN induction. The ability of the immune system to detect viral virulence factors was not limited to ICP0 from HSV-1. Ectopic expression of E40RF3, a functionally equivalent virulence factor from Adenovirus 5, induced a potent IFN response independently of PRR signaling molecules (FIG.
  • FIG. 7-a shows THPl and U937 cells, which also sensed both virulence factors to induce IFN.
  • Leaky doxycycline-independent expression of ICP0 was sufficient to induce IFN, whereas E40RF3 required doxy cy cline induction to be recognized (FIG. 7-a).
  • the enzymatic function of IPC0 is likely to be active at low concentrations, whereas E40RF3 polymerizes in order to sequester ND nuclear body components, which presumably requires higher expression levels. Consistent with its potent activity, ICP0 was hardly detected by immunoblot despite triggering a potent immune response (FIG. 8-b).
  • RNAseq was performed. Principal component analysis identified interferon-stimulated genes (ISGs) comprising PCI (FIG. 8-c) as the major discriminator between ICP0- and E40RF3 -expressing conditions and control cells (FIG. 1-e). Indeed, an enrichment analysis of genes with significantly increased transcription after virulence factor expression identified IFN signatures (FIG. 8-d) and multiple ISGs were activated strongly (FIG. 1-f). Co-clustering of virulence factor expressing conditions on the PCA plot indicated that the transcriptional response induced by ICP0 and E4ORF3 were overall comparable.
  • ICP0 displays SUMO-targeted ubiquitin ligase-like activity that marks sumoylated substrates for proteasomal degradation. Thus, its primary function is to degrade or inactivate host proteins.
  • ICP0 targets a negative regulator of IFN for degradation (FIG.2-a) inadvertently triggering an IFN response was considered. This hypothesis is consistent with the prior observation that genetic perturbance of the sumoylation machinery activates IFN in human monocytic cell lines.
  • a genome-wide CRISPR screen was conducted with the hope of identifying a nuclear, sumoylated substrate of ICP0 that negatively regulates IFN.
  • the screen relied on antibody staining of the cytosolic ISG-product Viperin to report IFN-status via autocrine signaling at single cell resolution (FIG.2-b).
  • Cells with spontaneous induction of Viperin were sorted from the bulk population (FIG.9-a) to enrich for sgRNAs whose targets are negative regulators of IFN under steady state conditions.
  • sgRNAs were validated to enhance Viperin expression at steady state with varying effect sizes (FIG.2-c), including sgRNAs targeting TRIM33, a known negative regulator of PRR-mediated IFN induction, and USP18, a known negative regulator of IFNAR signaling.
  • MORC3 sgRNAs targeting the sumoylated nuclear body protein MORC3 induced Viperin expression.
  • MORC3 was focused on as it was previously described to be degraded by ICP0 in fibroblasts. It was confirmed that ICP0 was required for MORC3 degradation during HSV-1 infection in BLaER1 monocytes (FIG.2-d) and U2OS cells, which support ICP0-independent HSV-1 replication (FIG.9-b). Independently generated MORC3 –/– BLaER1 monocytes (FIG.
  • FIG.9-c ⁇ FIG.9-g 2-e, FIG.9-c ⁇ FIG.9-g), or MORC3-targeted THP1 (FIG.10-a, FIG.10-b) and U937 (FIG.10-c, FIG.10-d) myeloid-like cells displayed spontaneous IFN induction.
  • MORC3-targeted THP1 FIG.10-a, FIG.10-b
  • U937 FIG.10-c, FIG.10-d
  • myeloid-like cells displayed spontaneous IFN induction.
  • the IFN response in MORC3 –/– BLaER1 cells did not require additional PRR activation and was independent of known PRR-signaling hubs TBK1/IKK ⁇ , IRF3/7 and IKK ⁇ / ⁇ (FIG.2-e, FIG.9-c ⁇ FIG.9-g).
  • MORC3 –/– monocytes recapitulated the transcriptional changes induced by virulence factors. This led to co-clustering of these conditions on the PCA plot (FIG.2-f), co-upregulation of ISGs by heatmap analysis (FIG.2-g), and enrichment of the same IFN-related modules in MORC3 –/– monocytes (FIG.9-h).
  • FIG.2-f co-clustering of these conditions on the PCA plot
  • FIG.2-g co-upregulation of ISGs by heatmap analysis
  • FIG.9-h enrichment of the same IFN-related modules in MORC3 –/– monocytes
  • MORC3 is a member of the MORC-gene family of GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPases that are transcriptional repressors. MORC3 has been proposed to bind H3K4me3 at promoters and participate in SUMO2-mediated transcriptional repression. It co-localizes with ND10 nuclear bodies and is required for p53 activation and senescence induction in response to genotoxic stress. In mice, MORC3 is embryonically lethal and partial loss of MORC3 impairs senescence in osteoclasts and induces their longevity, driving osteoporosis and related immune pathology.
  • GHKL gyrase, Hsp90, histidine kinase, MutL
  • MORC3 Localization of MORC3 to anti-viral ND10 nuclear bodies suggests a role for MORC3 during viral infection. Whereas one report observed enhanced viral replication in MORC3 knockdown cells (implying an anti-viral function of MORC3), other studies reported diminished viral replication in MORC3 knockdown cells and increased viral replication upon MORC3 overexpression (implying a pro-viral function of MORC3).
  • the primary function of MORC3 is apparent in cells that do not induce IFN upon MORC3 loss, such as HCT116 cells (FIG.10-e, FIG.10-f).
  • the primary anti-viral function of MORC3 may be guarded by its secondary IFN-repressing function.
  • These two functions are intimately linked such that viruses that express virulence factors to degrade MORC3, and avoid its primary anti-viral function, will unleash the secondary anti-viral interferon response (FIG.3-c).
  • FOG.3-c secondary anti-viral interferon response
  • MORC3 is not required to directly repress antiviral ISGs.
  • a small number of genes, including IFNB1 were still de-repressed in MORC3 –/– IFNAR1 –/– IFNAR2 –/– monocytes, which suggests they may be direct targets of MORC3 repression (FIG.3-d).
  • the most significantly de-repressed genes in MORC3 –/– IFNAR1 –/– IFNAR2 –/– monocytes are MLLT3, IFNB1 and FOCAD, which, remarkably, are clustered together within a short section of chromosome 9 (chr9:20,329,000-21,086,000; FIG.11-a).
  • a retroviral-based randomly integrated IFNB1-promoter-luciferase reporter was only triggered by DNA- STING signaling but was not activated in the absence of MORC3 (FIG.3-g).
  • the endogenous IFNB1 gene was activated both by STING signaling and MORC3 deficiency (FIG.3-h).
  • MORC3 participates in repression of endogenous retroviruses (ERVs). However, only a minimal de-repression of ERVs in MORC3 deficient monocytes (FIG.12-a) was found.
  • ERV repression likely occurs by distinct mechanisms in embryonic stem cells and monocytes, with MORC3-independent ERV repression mechanisms, such as DNA methylation, predominating in monocytes.
  • MORC3 acts to repress the IFNB1 locus rather than the IFNB1 promoter, whereas canonical PRR-mediated induction of IFNB1 is promoter-dependent and locus- independent.
  • ISG induction downstream of STING activation in BLaER1 monocytes did not rely solely on IFNB1, as revealed by IFNAR-dependent induction of ISGs, such as RSAD2, CXCL10, in an IFNB1- independent manner in response to foreign DNA (FIG.4-a, FIG.13-a).
  • BLaER1 monocytes are competent to make IFNs other than IFNB1.
  • IFNB1 is the only IFN-gene within the MORC3-repressed genomic region on chromosome 9 (FIG. 3-d) and is thus the only IFN-gene that is de-repressed in MORC3 –/– cells (FIG.4-b).
  • MORC3 acts narrowly and selectively on the IFNB1 locus and does not regulate other linked type I IFN genes.
  • activation of the MORC3 pathway by viral virulence factors uniquely relies on IFNB1 for anti-viral ISG induction (FIG.4-g).
  • MORC3-regulated element in an intron of FOCAD, approximately 100kb downstream of IFNB1, that gains DNA accessibility in the absence of MORC3 (FIG.14-a, FIG.14-b).
  • the MRE sequence is set forth as SEQ ID NO: 3.
  • the MRE was required for IFNB1 and MLLT3 de-repression in MORC3 –/– cells but not for IFNB1 induction upon PRR signaling, indicating that the MRE is a specific component of the MORC3 pathway (FIG.14-c, FIG.15-a, FIG.15-b).
  • MRE is located in an intron of FOCAD
  • FOCAD protein expression was not impaired in MRE ⁇ / ⁇ cells, and neither FOCAD nor MLLT3 were required for IFNB1 induction in absence of MORC3 (FIG.15-c ⁇ FIG.15-e). It was found that MRE regulates IFNB1 in cis because a MORC3-sgRNA drove bi-allelic expression in WT cells and mono-allelic expression of IFNB1 in heterozygous MRE +/ ⁇ cells (FIG.14-d, FIG.14-e).
  • MORC3 was targeted in primary human macrophages and a strong, spontaneous upregulation of IFNB1, MLLT3, and ISGs (FIG.15-h) was observed.
  • MORC3 represses a regulatory DNA element (MRE).
  • MRE regulatory DNA element
  • the MRE mediates activation of adjacent genes in cis which explains the location-specific de-repression of IFNB1.
  • mice harboring a conditional (floxed) allele of Morc3 were generated. These mice were then crossed to mice harboring an inducible CreERT2 gene.
  • MORC3-pathway as a novel innate immune sensing mechanism that detects the enzymatic activity of virulence factors from DNA viruses.
  • the MORC3 pathway induces a strong IFNB1 response redundantly with the cGAS-STING-pathway of cytosolic DNA sensing.
  • the STING-pathway is solely required for IFN induction during HSV-1 infection in other cell types, the MORC3-pathway might rely on monocyte-specific components that remain to be identified.
  • the MORC3-pathway does not utilize a PAMP- sensing receptor akin to a PRR; nor does it depend on canonical PRR signaling components that many viruses have evolved to disrupt or inhibit. Instead, it employs the self-guarded protein MORC3, whose bi-functionality allows detection of pathogen- encoded enzymatic activities.
  • the primary function of MORC3 appears to be to inhibit replication of HSV-1 and likely other (DNA) viruses that replicate in the nucleus, a function that is presumably connected to its association with ND10 nuclear bodies.
  • DNA viruses employ virulence factors, e.g. HSV-1 ICP0 and Ad5 E4ORF3, to inhibit ND10 nuclear bodies and/or degrade MORC3.
  • MORC3 may have evolved a secondary function: locus-specific repression of the IFNB1 gene. This secondary function allows activation of anti-viral IFN upon virulence factor- mediated perturbation of ND10 nuclear bodies and MORC3. De-repression of the IFNB1 locus may be less susceptible to pathogen interference or tumor cell evasion as compared to a multi-step PRR signaling pathway to activate IFNB1 transcription.
  • the MORC3 pathway might also explain prior observations of IRF3/7-independent IFN ⁇ during DNA virus infections and connections between perturbed sumoylation and IFN induction.
  • the integration of two distinct functions within a single ‘dead-man’s switch’ provides robust self-insurance of the anti-viral MORC3 function.
  • MORC3 exerts its two different functions by executing one unifying molecular activity, namely, transcriptional repression.
  • Various repressive activities have been proposed for the MORC gene family, including DNA methylation, H3K9-methylation, H3.3 incorporation and DNA compaction.
  • the identification of the MRE as a key downstream component required for IFNB1 expression in MORC3-deficient cells is interesting.
  • Loss of the MRE is the only known way to eliminate IFNB1 expression after activation of the MORC3 pathway, but importantly, loss of the MRE does not affect canonical IFNB1-inducing pathways that depend on IRF3/7. Thus, MRE mutant cells can be used to assess whether a specific intervention (e.g., MORC3 inhibitor) is affecting the MORC3 pathway.
  • MORC3 inhibitor e.g., MORC3 inhibitor
  • the MORC3 pathway is distinct from known PRR-mediated pathways of IFN induction, the strategy of self-guarded anti-viral proteins may be more common than has been previously appreciated.
  • MORC3 inhibits expression of the interferon beta gene
  • IFNB1 IFNB1
  • loss of MORC3 activity activates a Type I interferon induction pathway that is independent of pattern-recognition-receptors (PRRs).
  • PRRs pattern-recognition-receptors
  • the present invention provides methods for modulating an interferon response in a subject which comprises modifying the activity of MORC3 in the subject.
  • the activity of MORC3 is inhibited or reduced.
  • the activity of MORC3 is enhanced, increased, or stabilized.
  • MORC3 activity is modified in combination with administration of one or more immunomodulators in the art.
  • the activity of MORC3 in a subject may be inhibited or reduced by, e.g ., knock- out or knock-down of the MORC3 gene in the subject, administering the subject an siRNA that inhibits or reduces expression of MORC3, administering the subject a MORC3 inhibitor, etc.
  • the activity of MORC3 in a subject may be enhanced or increased by, e.g. , increasing the copy number of MORC3 in the subject, administering MORC3 to the subject, etc.
  • MORC3 activity is modified in a subject in need thereof.
  • a subject “in need of’ modification of MORC3 activity is one who will likely benefit from an increase or decrease of interferon.
  • the subject is one who suffers from an abnormality in an interferon induction signaling pathway, e.g. , RIG-I pathway, TRIF pathway, and IRF7 pathway.
  • the subject is in need of an increase of interferon.
  • the subject is in need of a decrease of interferon.
  • MORC3 activity is inhibited or reduced:
  • MORC3 activity is enhanced, increased, or stabilized to treat an inflammatory disease or disorder involving IFNB1 expression, a disease caused by hyper-IFN expression, systemic lupus erythematosus (SLE), rheumatoid arthritis, multiple sclerosis (MS), type I diabetes (T1D), and Sjogren’s syndrome, myositis, etc.
  • SLE systemic lupus erythematosus
  • MS multiple sclerosis
  • T1D type I diabetes
  • Sjogren’s syndrome myositis, etc.
  • MORC3 activity is enhanced, increased, or stabilized to treat a bacterial or viral infection.
  • Methods in the art may be used to identify small molecule inhibitors of MORC3 expression and/or activity.
  • the small molecule inhibitors block the ATPase activity of MORC3.
  • cells lacking the signaling components such as
  • TBK1/IKKE, IKKa/b, and/or IRF3/7 are treated with a candidate compound to determine whether the candidate compound stimulates or inhibits interferon production, and thereby indicates whether the candidate compound modulates MORC3 activity.
  • a compound that modulates MORC3 activity may be optimized to improve its pharmacokinetics using methods in the art and/or targeted to a tissue of interest, e.g. , conjugated to a carrier molecule.
  • an “interferon disorder” refers to a disease, infection, or disorder that results in and/or involves an abnormally high or an abnormally low amount of interferon production.
  • the interferon disorder is an IFNB1 Disorder.
  • an “IFNB1 Disorder” refers to diseases and disorders that are, directly or indirectly, caused by abnormal interferon beta (IFNB1) activity, e.g. , abnormally high or abnormally low levels of interferon as compared to normal control subjects.
  • IFN Excess Disorder refers to diseases and disorders that, directly or indirectly, cause or result in abnormally high levels of interferon as compared to normal control subjects. IFN Excess Disorders also include abnormally high levels of interferon resulting from infections and therapeutic interventions. Examples of IFN Excess Disorders include autoimmune and inflammatory diseases such as rheumatoid arthritis, psoriasis, vitiligo, hypothyroidism, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, myasthenia gravis,
  • autoimmune and inflammatory diseases include multiple sclerosis, primary progressive multiple sclerosis, and relapsing-remitting multiple sclerosis.
  • IFN Deficiency Disorder refers to diseases and disorders that, directly or indirectly, cause or result in abnormally low levels of interferon as compared to normal control subjects; and diseases, infections, and/or symptoms that are treatable by administration of interferon. IFN Deficiency Disorders also include abnormally low levels of interferon resulting from infections and therapeutic interventions.
  • IFN Deficiency Disorders include viral infections (e.g ., as herpes, hepatitis, a coronavirus infection (e.g., COVID 19), etc.), cancers (e.g, leukemias, lymphomas, melanomas, sarcomas, adenocarcinomas, etc.), and the like.
  • viral infections e.g ., as herpes, hepatitis, a coronavirus infection (e.g., COVID 19), etc.
  • cancers e.g, leukemias, lymphomas, melanomas, sarcomas, adenocarcinomas, etc.
  • MORC3 therapeutic agents refer to compounds and biomolecules that inhibit, reduce, increase, enhance, or stabilize MORC3 expression and/or activity.
  • MORC3 therapeutic agents include MORC3 inhibitors (e.g, siRNAs, small molecules, ATPase inhibitors (e.g, small molecule ATPase inhibitors), etc.) and MORC3 activators.
  • MORC3 inhibitors e.g, siRNAs, small molecules, ATPase inhibitors (e.g, small molecule ATPase inhibitors), etc.
  • MORC3 activators e.g, siRNAs, small molecules, ATPase inhibitors (e.g, small molecule ATPase inhibitors), etc.
  • MORC3 activators e.g, MORC3 activators.
  • the MORC3 therapeutic agent is a small molecule, a protein, or a nucleic acid.
  • the MORC3 therapeutic agent is an antibody, fusion protein, degrader, shRNA, siRNA, microRNA, asymmetric interfering RNA, antisense molecule, lhRNA, miRNA embedded shRNA, or small internally segmented RNA.
  • the MORC3 therapeutic agent is a MORC3 protein or a MORC3 fusion protein.
  • the MORC3 therapeutic agent comprises an ICP0 protein.
  • the MORC3 therapeutic agent comprises shRNA, siRNA, microRNA, asymmetric interfering RNA, antisense molecule, lhRNA, miRNA embedded shRNA, or small internally segmented RNA.
  • the MORC3 therapeutic agent is an siRNA, an ATPase inhibitor (e.g, a small molecule ATPase inhibitor), a MORC3 protein having at least about 90% sequence identity to SEQ ID NO: 1, or an ICP0 protein having at least about 95% sequence identity to SEQ ID NO: 2.
  • ATPase inhibitor e.g, a small molecule ATPase inhibitor
  • MORC3 protein having at least about 90% sequence identity to SEQ ID NO: 1 e.g., a small molecule ATPase inhibitor
  • ICP0 protein having at least about 95% sequence identity to SEQ ID NO: 2.
  • MORC3 inhibitors and activators include those that bind MORC3 and thereby results in the autoinhibited conformation and the activated conformation, respectively. See, e.g., Zhang etal. (2019) PNAS USA 116(13):6111- 6119.
  • the ATPase inhibitor is (-)-Blebbistatin, Bafilomycin A1 (Baf-Al), BHQ, Brefeldin A, BRITE338733, BTB06584, Bufalin, CB-5083, Dexlansoprazole, Digoxin (NSC 95100), Ilaprazole, Ilaprazole sodium, KM91104, Lansoprazole, ML241 hydrochloride, ML367, NEXIUM (esomeprazole magnesium), Oleandrin (PBI-05204), Omeprazole, Ouabain, Pantoprazole, Pantoprazole sodium,
  • the siRNA is selected from the group consisting of hsa-miR-3148, hsa-miR-144-3p, hsa- miR-101-3p, hsa-miR-9902, hsa-miR-367-3p, hsa-miR-3059-3p, hsa-miR-92b-3p, hsa- miR-25-3p, hsa-miR-92a-3p, hsa-miR-363-3p, hsa-miR-32-5p, hsa-miR-570-5p, hsa- miR-548ai, hsa-miR-548ba, hsa-miR-548ag, h
  • sample is used in its broadest sense and includes specimens and cultures obtained from any source, as well as biological samples and environmental samples.
  • Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases.
  • Biological samples include blood products, such as plasma, serum, and the like.
  • a biological sample can be obtained from a subject using methods in the art.
  • a sample to be analyzed using one or more methods described herein can be either an initial unprocessed sample taken from a subject or a subsequently processed, e.g. , partially purified, diluted, concentrated, fluidized, pretreated with a reagent (e.g., protease inhibitor, anti -coagulant, etc.), and the like.
  • a reagent e.g., protease inhibitor, anti -coagulant, etc.
  • the sample is a blood sample.
  • the blood sample is a whole blood sample, a serum sample, or a plasma sample.
  • the sample may be processed, e.g, condensed, diluted, partially purified, and the like.
  • the sample is pretreated with a reagent, e.g, a protease inhibitor.
  • two or more samples are collected at different time intervals to assess any difference in the amount of the analyte of interest, the progression of a disease or disorder, or the efficacy of a treatment.
  • the test sample is then contacted with a capture reagent and, if the analyte is present, a conjugate between the analyte and the capture reagent is formed and is detected and/or measured with a detection reagent.
  • kits for assaying MORC3, in a sample e.g, a biological sample from a subject.
  • the kits comprise one or more reagents, e.g, blocking buffers, assay buffers, diluents, wash solutions, etc., for assaying the MORC3.
  • the kits comprise additional components such as interpretive information, control samples, reference levels, and standards.
  • kits comprising one or more MORC3 therapeutic agents, optionally in a composition or in combination with one or more supplementary agents, packaged together with one or more reagents or drug delivery devices for preventing, inhibiting, reducing, or treating interferon disorder in a subject.
  • the kits comprise the one or more MORC3 therapeutic agents, optionally in one or more unit dosage forms, packaged together as a pack and/or in drug delivery device, e.g, a pre-filled syringe.
  • kits include a carrier, package, or container that may be compartmentalized to receive one or more containers, such as vials, tubes, and the like.
  • the kits optionally include an identifying description or label or instructions relating to its use.
  • the kits include information prescribed by a governmental agency that regulates the manufacture, use, or sale of compounds and compositions as contemplated herein.
  • the methods and kits as contemplated herein may be used in the evaluation of an interferon disorder, such as a IFN Deficiency Disorder or a IFN Excess Disorder.
  • the methods and kits may be used to monitor the progress of such a disease, assess the efficacy of a treatment for the disease, and/or identify patients suitable for a given treatment in a subject.
  • the methods and kits may be used to diagnose a subject as having a interferon disorder and/or provide the subject with a prognosis.
  • the methods and kits may be used to determine whether a subject exhibits a level of MORC3 that is low or high as compared to a control.
  • the control is a sample from a normal, healthy subject.
  • the control is a pooled sample from a plurality of normal, healthy subjects.
  • the control is a given reference level. The abnormal level may then be used to diagnose the subject as suffering from an interferon disorder.
  • a subject identified as having a low level or a high level of MORC3 may be subjected to a suitable treatment.
  • a subject identified as having a high level of MORC3 or diagnosed as suffering from an IFN Excess Disorder may be treated with one or more MORC3 therapeutic agents.
  • a subject identified as having a low level of MORC3 or diagnosed as suffering from a IFN Deficiency Disorder may be treated with one or more MORC3 therapeutic agents.
  • the methods and kits may be used to monitor the efficacy of treatment with a given therapeutic, e.g ., a MORC3 therapeutic agent, that modulates the level of MORC3 produced in the subject and the dosage of the given therapeutic may be adjusted accordingly.
  • a given therapeutic e.g ., a MORC3 therapeutic agent
  • the methods and kits may be used for research purposes.
  • the methods and kits may be used to identify diseases that are caused by abnormal levels of MORC3 and/or identify diseases that result in abnormal levels of MORC3.
  • the methods and kits may be used to study mechanisms, e.g. , mechanisms and pathways involving MORC3.
  • the methods and kits may be used to develop and screen for therapeutics that increase or decrease levels of MORC3 in subjects.
  • compositions comprising, consisting essentially of, or consisting of one or more MORC3 therapeutic agents are contemplated herein.
  • pharmaceutical composition refers to a composition suitable for pharmaceutical use in a subject.
  • a composition generally comprises an effective amount of an active agent and a diluent and/or carrier.
  • a pharmaceutical composition generally comprises a therapeutically effective amount of an active agent and a pharmaceutically acceptable carrier.
  • pharmaceutical compositions may include one or more supplementary agents. Examples of suitable supplementary agents include immunomodulatory agents, interferon, and the like.
  • an “effective amount” refers to a dosage or amount sufficient to produce a desired result.
  • the desired result may comprise an objective or subjective change as compared to a control in, for example, in vitro assays, and other laboratory experiments.
  • a “therapeutically effective amount” refers to an amount that may be used to treat, prevent, or inhibit a given disease or condition in a subject as compared to a control, such as a placebo.
  • a control such as a placebo.
  • the one or more MORC3 therapeutic agents may be administered, preferably in the form of pharmaceutical compositions, to a subject.
  • the subject is mammalian, more preferably, the subject is human.
  • Preferred pharmaceutical compositions are those comprising at least one MORC3 therapeutic agent in a therapeutically effective amount and a pharmaceutically acceptable vehicle.
  • treatment of a subject with a therapeutically effective amount may be administered as a single dose or as a series of several doses.
  • the dosages used for treatment may increase or decrease over the course of a given treatment.
  • Optimal dosages for a given set of conditions may be ascertained by those skilled in the art using dosage-determination tests and/or diagnostic assays in the art. Dosage-determination tests and/or diagnostic assays may be used to monitor and adjust dosages during the course of treatment.
  • compositions may be formulated for the intended route of delivery, including intravenous, intramuscular, intra peritoneal, subcutaneous, intraocular, intrathecal, intraarticular, intrasynovial, cisternal, intrahepatic, intralesional injection, intracranial injection, infusion, and/or inhaled routes of administration using methods known in the art.
  • compositions may include one or more of the following: pH buffered solutions, adjuvants (e.g ., preservatives, wetting agents, emulsifying agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • adjuvants e.g ., preservatives, wetting agents, emulsifying agents, and dispersing agents
  • liposomal formulations e.g., nanoparticles, dispersions, suspensions, or emulsions
  • sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • compositions may be administered to a subject by any suitable route including oral, transdermal, subcutaneous, intranasal, inhalation, intramuscular, and intravascular administration. It will be appreciated that the preferred route of administration and pharmaceutical formulation will vary with the condition and age of the subject, the nature of the condition to be treated, the therapeutic effect desired, and the particular MORC3 therapeutic agent used.
  • a “pharmaceutically acceptable vehicle” or “pharmaceutically acceptable carrier” are used interchangeably and refer to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration and comply with the applicable standards and regulations, e.g., the pharmacopeial standards set forth in the United States Pharmacopeia and the National Formulary (USP-NF) book, for pharmaceutical administration.
  • UDP-NF National Formulary
  • unsterile water is excluded as a pharmaceutically acceptable carrier for, at least, intravenous administration.
  • Pharmaceutically acceptable vehicles include those known in the art. See, e.g, Remington: The Science and Practice of Pharmacy 20th ed (2000) Lippincott Williams & Wilkins, Baltimore, MD.
  • the pharmaceutical compositions may be provided in dosage unit forms.
  • a “dosage unit form” refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the one or more MORC3 therapeutic agent calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the given MORC3 therapeutic agent and desired therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of MORC3 therapeutic agents according to the instant invention and compositions thereof can be determined using cell cultures and/or experimental animals and pharmaceutical procedures in the art.
  • LCso the dose expressed as concentration x exposure time that is lethal to 50% of the population
  • LDso the dose lethal to 50% of the population
  • EDso the dose therapeutically effective in 50% of the population
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • MORC3 therapeutic agents which exhibit large therapeutic indices are preferred. While MORC3 therapeutic agents that result in toxic side-effects may be used, care should be taken to design a delivery system that targets such compounds to the site of treatment to minimize potential damage to uninfected cells and, thereby, reduce side-effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans.
  • Preferred dosages provide a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • Therapeutically effective amounts and dosages of one or more MORC3 therapeutic agents can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a dosage suitable for a given subject can be determined by an attending physician or qualified medical practitioner, based on various clinical factors.
  • BLaERl, E1937 and THP-1 cells were cultured in RPMI Medium 1640 supplemented with L-glutamine, sodium pyruvate, 100 El/ml penicillin-streptomycin (Thermo Fisher) and 10% (v/v) FCS (Omega Scientific).
  • HEK293T, U20S and HCT116 cells were cultivated in DMEM Medium (Thermo Fisher) containing the same supplements.
  • 1.4 million BLaERl cells per well of a 6-well plate were trans- differentiated into monocytes for 5-6 days in medium containing 10 ng/ml of hrIL-3, 10 ng/ml hr-CSF-1 (M-CSF) (both PeproTech) and 100 nM ⁇ -Estradiol (Sigma-Aldrich) as previously described.
  • 1.4 million THP-1 and U937 cells per well of a 6-well plate were differentiated overnight with 100 ng/ml PMA (Sigma-Aldrich). STING-deficient and corresponding control THP1 and U937 cells were a gift from Dan Stetson (University of Washington).
  • BLaER1 cells were a gift from Thomas Graf (CRG, Barcelona, Spain) and Veit Hornung (LMU Kunststoff, Germany).
  • U2OS cells were a gift from Robert Tjian and Xavier Darzacq (UC Berkeley).
  • THP1 cells were from ATCC.
  • U937 cells were from the UC Berkeley Cell Culture Facility.
  • HCT116 cells were a gift from David Raulet (UC Berkeley).
  • doxycycline-inducible trans-gene expression cells were stimulated with 1 ⁇ g/ml doxycycline hyclate (Sigma-Aldrich) for 24 h.
  • HSV-1 infection [0099] BACs of ⁇ ICP0 HSV-1 and corresponding WT strain were a gift from Bernard Roizman (University of Chicago). BAC DNA was prepared from a mono-clonal transformant and transfected into U2OS cells using Lipofectamine 2000 (Thermo Fisher). Virus was propagated, harvested and frozen as described. Viral progeny were titered from cell-free supernatants by TCID50 using 8 replicates per dilution.
  • U2OS cells were used for titering if not otherwise indicated, and FFU/ml was calculated by the Spearman & Kärber algorithm.
  • Myeloid cells were infected by adsorbing virus of appropriate MOI in FCS free RPMI Medium 1640 for 1 h. Subsequently, medium was changed to complete RPMI Medium 1640. For analysis of viral progeny in the supernatant, cells were subsequently washed three times with warm PBS and resuspended in RPMI. For all other experiments, medium was directly changed to RPMI without a PBS wash.
  • RT-qPCR reverse transcription
  • RSAD2.fwd (SEQ ID NO: 4): CAACTACAAATGCGGCTTCT RSAD2.rev (SEQ ID NO: 5): ATCTTCTCCATACCAGCTTCC CXCL10.fwd (SEQ ID NO: 6): TCTGAATCCAGAATCGAAGG CXCL10.rev (SEQ ID NO: 7): CTCTGTGTGGTCCATCCTTG GAPDH.fwd (SEQ ID NO: 8): GAGTCAACGGATTTGGTCGT GAPDH.rev (SEQ ID NO: 9): GACAAGCTTCCCGTTCTCAG IFNB1.fwd (SEQ ID NO: 10): CAGCATCTGCTGGTTGAAGA IFNB1.rev (SEQ ID NO: 11): CATTACCTGAAGGCCAAGGA MLLT3.fwd (SEQ ID NO: 12): GAGCACAGTAACATACAGCA MLLT3.rev (SEQ ID NO: 13): GGCAAAATGAA
  • Laemmli buffer was added to a final concentration of 1 x and lysates were boiled at 95 °C for 10 minutes. Proteins were separated with denaturing PAGE and transferred to Immobilon- FL PVDF membranes (Millipore Sigma). Membranes were blocked with Li-Cor Odyssey blocking buffer. Primary antibodies were added and immunoblots incubated overnight.
  • IFNB1 coding sequence with distinct indels were engineered. This enabled us to track which allele is being transcribed upon activation of IFNB1 by amplicon sequencing of IFNB1 cDNA (Fig.4d). Cytosolic DNA sensing drove bi-allelic activation of IFNB1 regardless of the MRE. Activating the MORC3 pathway with a MORC3-sgRNA drove bi-allelic expression in WT cells and mono-allelic expression of IFNB1 in heterozygous MRE+/' cells.
  • Monoclonal gene deficient BLaERl cells were generated as follows. Briefly, sgRNAs specific for the indicated genes, were designed to target an early coding exon of the respective gene with minimal off-targets and high on-target activity using ChopChop. U6-sgRNA-CMV-mCherry-T2A-Cas9 plasmids were generated by ligation-independent- cloning as previously described and BlaERl cells were electroporated using a Biorad GenePulser device. Automated cell sorting was used to collect mCherry positive cells that were cloned by limiting dilution. Monoclonal cell lines were identified, rearranged and duplicated for genotyping using deep sequencing as previously described.
  • Knockout cell clones contained all-allelic frame shift mutations without any wild type reads. Two independent knockout single-cell clones were analyzed per genotype, and one representative clone per genotype is shown. For polyclonal gene targeting, cell lines were transduced with lentiCas9-Blast, a gift from Feng Zhang (Addgene plasmid # 52962; http://n2t.net/addgene:52962; RRID: Addgene_52962).
  • sgRNAs were designed as above and cloned into lentiGuide-Puro, a gift from Feng Zhang (Addgene plasmid # 52963; http://n2t.net/addgene:52963; RRID: Addgene_52963), using ligation- independent-cloning. Cas9-expressing cells were transduced with indicated sgRNA- encoding lenti-viruses.
  • Lenti-/retro-viral transduction [0112] Lenti- and retro-virus was produced in HEK293T cells. 4.5 million cells were plated per 10 cm dish and transfected with 5 ⁇ g of transfer vector, 3.75 ⁇ g of packaging vector (pd8.9 for lenti- and pGAGPOL for retro-virus) and 1.5 ⁇ g pVSVG using 30.75 ⁇ g PEI-MAX (Polysciences, 24765-1). 12 h after transfection the medium was replaced with DMEM medium containing 30% (v/v) FCS. After 24-36 h, viral supernatants were harvested, centrifugated at 1000 x g for 10 min, and filtered through a 0.45 ⁇ m filter. Cells were culture for 48 h after transduction, prior to selection with puromycin or Blasticidine S hydrochloride (both Sigma-Aldrich). [0113] Ectopic gene expression
  • a doxycycline-inducible lenti -virus system was used for ectopic gene expression as previously described. Codon-optimized constructs for HSV-1-ICP0 and HA- Adenovirus5-E40RF3 were synthesized by Integrated DNA Technologies and cloned into pLIP. ICPO variants were generated by overlap-extension PCR.
  • the 1 kb upstream of the transcription start site of huma IFNB1 (hg38 chr9:21077923-21078922) was synthesized by Integrated DNA Technologies and cloned in front of a luciferase reporter from Gaussia princeps into a retro-viral transfer vector in opposite direction to the 5’LTR. BLaERl cells were transduced and sorted for reporter integration.
  • BLaERl were harvested for flow cytometry, fixed and permeabilized using eBioscienceTM IC Fixation Buffer and eBioscienceTM Permeabilization Buffer (both Thermo Fisher) according to the provider’s protocol.
  • Cells were incubated for 1 h with PE-Anti-Viperin (Clone MaP.VIP; BD, 565196) and analyzed using a BD LSRFortessaTM Flow Cytometer. If indicated, cells were sorted on a BD FACSAriaTM Fusion Cell Sorter.
  • HSV-1 stain buffer PBS, 10% FCS, 1 mM EDTA and 0.1% saponin
  • BD Human BD Fc Block
  • HSV-1 staining buffer After 3 washes with HSV-1 staining buffer, cells were stained with anti-rabbit secondary antibody (A-21244, Thermo Fisher Scientific) at 1:2,000 dilution in HSV-1 stain buffer, washed 4 times in HSV-1 stain buffer and analysed using a BD LSRFortessa Flow Cytometer.
  • sgRNA positive cells were sorted irrespectively of their Viperin expression. The reactions were incubated at 65 °C for 10 min and at 95 °C for 15 min. The integrated sgRNA cassette was amplified using a nested PCR approach with Phusion DNA Polymerase (Thermo Fisher) with 4 technical replicates per biological replicate. Primers for the first level utilized a mix of staggered forward primers: pMCB320fwd Ont stagger (SEQ ID NO: 41):
  • RNA-Seq RNA-Seq
  • RNA from 2.8 million trans-differentiated BLaERl monocytes was isolated using
  • RNA-seq libraries were prepared by the QB3 Genomics Functional Genomics Laboratory from poly-A-enriched mRNA using a KAPA mRNA HyperPrep Kit (Roche) and sequenced on the Illumina Novaseq S4 150PE.
  • RNA-seq reads were aligned to the reference genome (GRCh38.83) using Bowtie2 with default settings.
  • Transcript and gene counts were quantified using RSEM with the parameter ‘strandedness’ set to ‘reverse’ to account for strand-specific library preparation protocol.
  • DeSeq2 was used to identify differentially expressed genes between conditions by building a single DeSeq2 model using counts from RSEM and performed pairwise comparisons of conditions. All genes with a false discovery rate (FDR) below 0.05 were considered to be significantly differential.
  • Log normalized counts from DeSeq2 were used to perform principal component analysis (PCA) shown in FIG. 2-a - FIG. 2-g and to generate heatmaps.
  • PCA principal component analysis
  • Gene enrichment analysis was done with the R package clusterProfiler. For each enrichment analysis, the foreground was assigned to be the set of genes with significant increased expression in the condition being evaluated. The background was set to the set of all genes that have mean counts greater than 1 for the condition being evaluated. Gene enrichment sets were downloaded from the Molecular Signatures Database (MSigDB).
  • MSigDB Molecular Signatures Database
  • ATAC-seq was performed using methods in the art. 50,000 BLaERl monocytes were washed with PBS and lysed in 50(pl of cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgC12, 0.1% IGEPAL CA-630). Nuclei were collected by centrifugation and resuspended in 50(m1 TD Buffer (Illumina, FC-121-1030) with 2.5 m ⁇ Tn5 Transposase (Illumina, FC-121-1030). The reaction was incubated for 30(min at 37(°C and DNA was isolated with a MinElute Kit (Qiagen). Transposed DNA fragments were amplified to reach 30% of maximal amplification using Adi and Ad2 primer and sequenced on an Illumina Nova-Seq SP 50PE.
  • cold lysis buffer 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgC12, 0.
  • CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGT Ad2.4 (SEQ ID NO: 54):
  • CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGT Ad2.5 (SEQ ID NO: 55):
  • FASTQC version 0.11.539 was used to assess quality of ATAC-seq reads. Adapter sequences were removed using Cutadapt version 2.1046 using a default error rate of 0.1. Reads shorter than 5 were discarded. Reads were aligned to the hgl9 reference genome using bowtie 2 version 2.3.240 and discordant alignments were removed. Reads with mapping quality less than 30 were removed using SAMtools version 1.3.147 and duplicates were removed using Picard Tools version 2.5.0 (broadinstitute.github.io/picard/). Additionally, regions overlapping black list regions, identified from the ENCODE consortium, were removed. Blacklist regions were downloaded from ENCODE (accession ENCFF000KJP).
  • ATAC-seq reads aligned to the positive strand were shifted +4 bp and reads aligned to the negative strand were shifted -5 bp to adjust read start sites to represent the center of the transposase-binding event.
  • Peaks were called on shifted reads using MACS2 version 2.2.7.150, setting the FDR to 0.05 and the default human genome size. Peaks from all samples were combined by taking the union of all peaks. Counts representing peak strength for all samples were obtained by counting the number of cut sites that overlapped each peak for each sample. DeSeq2 version 1.30.142 was used to identify the differential abundance of cut sites between conditions. All regions with an FDR below 0.05 were marked as significantly differentially accessible.
  • RNA-seq reads from IFNAR1 –/– IFNAR2 –/– mCherry and MORC3 –/– IFNAR1 –/– IFNAR2 –/– BLaER1 monocytes were aligned to the hg38 genome using the STAR aligner with parameter ‘outFilterMultimapNmax’ set to ‘100000000’ to allow for lenient alignment of multimapped reads.
  • RepEnrich2 was used to quantify expression of ERV families.
  • the MAPQ threshold for subsetting uniquely mapping and multi-mapping reads in RepEnrich2 was set to 255.
  • the pre-built repeat annotations for RepEnrich2, available through the RepEnrich2 GitHub was used.
  • Differential expression of ERV counts from RepEnrich2 was analyzed using both the recommended edgeR and DeSeq2 pipelines to identify an overlapping set of high confidence differentially expressed ERVs. All ERVs with an FDR below 0.05 were considered to be significantly differential.
  • PBMCs Human peripheral blood mononuclear cells
  • AllCells Almeda
  • Alpha IRB approval obtained by AllCells (7000-SOP-045) for the study ‘Non-Mobilized Mononuclear Cell Apheresis Collection from Healthy Donors for the Research Market’.
  • Monocytes were isolated by negative selection (Pan Monocyte Isolation Kit, Miltenyi Biotec) and nucleofected with Cas9 gRNA ribonucleoparticles as previously described55.
  • Alt-R CRISPR RNAs crRNAs
  • tracrRNA Alt-trans-activating CRISPR RNA
  • IDT Alt-trans-activating CRISPR RNA
  • crRNA target sites MORC3 (SEQ ID NO: 57): GCTGATACTGAGATACCATATGG scramble (SEQ ID NO: 58): GCTGCTCCCTAACAGGACGC [0133] Assays for MORC3 Inhibitors that Induce an IFN Response [0134] The following assays may be used to screen for MORC3 inhibitors that induce an IFN response: [0135] BLaER1 human monocytes: Transdifferentiated 70,000 BLaER1 human monocytes are cultivated in RPMI 1640 Medium supplemented with 2 mM L-Glutamine, 1 mM Sodium Pyruvate, 100 U/mL Penicillin, 100 U/mL Streptomycin, 10% (v/v) heat- inactivated FCS, 10 ng/mL hrIL-3, 10 ng/mL hr-M-CSF and 100 nM Estradiol for 5 days at 37 °C and 5% CO 2 .
  • the culture media is change to plain RPMI without hrIL-3, hr-M- CSF, and Estradiol. Then a given candidate MORC3 inhibitor is added thereto and incubated for about 24-48 hours at 37 °C and 5% CO2. Cell-free supernatant is harvested therefrom and an IFNB1 ELISA (R&D, DY814-05) is performed according to the manufacturer’s protocol and Type I IFN Bioassay (HEK-BlueTM IFN- ⁇ / ⁇ Cells, Invivogen, hkb-ifnab) against a recombinant protein standard as recommended by the provider and the results are compared to a control.
  • IFNB1 ELISA R&D, DY814-05
  • Type I IFN Bioassay HEK-BlueTM IFN- ⁇ / ⁇ Cells, Invivogen, hkb-ifnab
  • Gene-deficient BLaER1 monocytes may be used to validate that the IFN response induced by a candidate MORC3 inhibitor mirrors the IFN response induced by a genetic MORC3 deficiency.
  • Primary human monocytes PBMCs are isolated from peripheral blood of human donors and overlaid with 13 ml of Ficoll solution (Millipore Sigma, GE17-1440-02) and 25 ml of anti-coagulated peripheral blood.
  • the cells are pelletized, e.g., at about 2000 rpm for 20 minutes at 22°C to result in a pellet of and a white disc on top of the ficoll layer.
  • the disc is aspirated from slightly above with a 10 ml pipet and transferred to a new tube. NaCl is added thereto to give a final volume of 50 ml.
  • the mixture is spun at 450 rcf for 7 min.
  • the pellet is resuspended in 10 ml of erythrocyte lysis solution (e.g., 1 mL BD Pharm Lysis concentrate and 9 ml H 2 O). Cells are lysed for 5 min. Then NaCl is added to give a final volume of 50 ml.
  • CD14+ monocytes may be optionally isolated using CD14 MicroBeads, human (Miltenyi, 130-050-201).
  • the cells are plated and the given candidate MORC3 inhibitor is added thereto and then incubated for 24-48 h at 37 °C and 5% CO2.
  • the cell-free supernatant is harvested and an IFNB1 ELISA (R&D, DY814- 05) is performed according to the manufacturer’s protocol and Type I IFN Bioassay (HEK-BlueTM IFN- ⁇ / ⁇ Cells, InvivoGen, hkb-ifnab) against a recombinant protein standard as recommended by the provider and the results are compared to a control.
  • Assay for Assessing the ATPase Activity of MORC3 The following is an exemplary protocol for assessing the ATPase activity of MORC3: 1) PCR amplify human MORC3, including the full-length open reading frame, or the ATPase and CW domains, or the ATPase only domain. 2) Clone each PCR product into pET28a such that it has a N-Terminal 6xHis Tag attached by a Thrombin site to Morc3. 3) After validating each construct by sequencing, transform into BL21 RIL cells. Inoculate a single colony in 2 mL LB with Kanamycin at a final concentration of 50 ⁇ g/ml.
  • ii) Resuspend in 30 mL Lysis Buffer (20 mM Tris pH 8.0, 400 mM NaCl, 1% Triton) in a fresh 50 mL conical tube.
  • iii) Add 10 ⁇ L DNase I (Thermo Cat EN0525) and 10 ⁇ L Benzonase (Sigma Cat# E1014).
  • iv) Add 2 mM MgCl 2 .
  • Sonicate sample vi) Spin sample at 12,000 x g for 15 min. vii) Transfer supernatant to a fresh tube. viii) Add binding agents.
  • MORC3 a Component of PML Nuclear Bodies, Has a Role in Restricting Herpes Simplex Virus 1 and Human Cytomegalovirus. J Virol 90, 8621-8633, doi:10.1128/JVI.00621-16 (2016). Van de Weyer, A. L. et al. A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana. Cell 178, 1260-1272 e1214, doi:10.1016/j.cell.2019.07.038 (2019). Boyden, E. D. & Dietrich, W. F. Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin.
  • Rho GTPases Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1. Nature 496, 233-237, doi:10.1038/nature12025 (2013).
  • Xu, H. et al. Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome. Nature 513, 237-241, doi:10.1038/nature13449 (2014). Luecke, S. & Paludan, S. R. Innate recognition of alphaherpesvirus DNA.
  • PML contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by ICP0.
  • Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100.
  • C/EBPalpha induces highly efficient macrophage transdifferentiation of B lymphoma and leukemia cell lines and impairs their tumorigenicity.
  • Fitzgerald, K. A. et al. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol 4, 491-496, doi:10.1038/ni921 (2003). Sato, M. et al. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction.
  • NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression.
  • Takahashi, K. et al Dynamic regulation of p53 subnuclear localization and senescence by MORC3.
  • clusterProfiler an R package for comparing biological themes among gene clusters. OMICS 16, 284-287, doi:10.1089/omi.2011.0118 (2012). Liberzon, A. et al. Molecular signatures database (MSigDB) 3.0. Bioinformatics 27, 1739-1740, doi:10.1093/bioinformatics/btr260 (2011). Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21, doi:10.1093/bioinformatics/bts635 (2013). Criscione, S. W., Zhang, Y., Thompson, W., Sedivy, J. M. & Neretti, N.
  • a human MORC3 comprises at least 90% sequence identity to Accession No. NP_001307374 or NP_056173.1. In some embodiments, a human MORC3 comprises at least 95% sequence identity to Accession No. NP_001307374 or NP_056173.1. In some embodiments, a human MORC3 comprises at least 97% sequence identity to Accession No. NP_001307374 or NP_056173.1. In some embodiments, the sequence of a human MORC3 is NP_001307374.1 or NP_056173.1.
  • a “MORC3 protein” refers to a protein having at least 90% sequence identity to SEQ ID NO: 1.
  • an “ICP0 protein” refers to a protein having at least about 95% sequence identity to SEQ ID NO: 2.
  • the terms “subject”, “patient”, and “individual” are used interchangeably to refer to humans and non-human animals.
  • the terms “non-human animal” and “animal” refer to all non-human vertebrates, e.g., non-human mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects and test animals.
  • the subject is a mammal.
  • the subject is a human.
  • diagnosis refers to the physical and active step of informing, i.e., communicating verbally or by writing (on, e.g, paper or electronic media), another party, e.g. , a patient, of the diagnosis.
  • prognosis refers to the physical and active step of informing, /. e. , communicating verbally or by writing (on, e.g. , paper or electronic media), another party, e.g. , a patient, of the prognosis.
  • A, B, or both A and B” and “A, B, C, and/or D” means “A, B, C, D, or a combination thereof’ and said “A, B, C, D, or a combination thereof’ means any subset of A, B, C, and D, for example, a single member subset (e.g, A or B or C or D), a two-member subset (e.g, A and B; A and C; etc.), or a three-member subset (e.g, A, B, and C; or A, B, and D; etc.), or all four members (e.g, A, B, C, and D).
  • a single member subset e.g, A or B or C or D
  • a two-member subset e.g, A and B; A and C; etc.
  • a three-member subset e.g, A, B, and C; or A, B, and D; etc.
  • all four members e.g, A, B, C
  • C means “one or more of A”, “one or more of B”, “one or more of C”, “one or more of A and one or more of B”, “one or more of B and one or more of C”, “one or more of A and one or more of C” and “one or more of A, one or more of B, and one or more of C”.
  • the phrase “consists essentially of’ in the context of a given ingredient in a composition means that the composition may include additional ingredients so long as the additional ingredients do not adversely impact the activity, e.g, biological or pharmaceutical function, of the given ingredient.
  • composition comprises, consists essentially of, or consists of A.
  • the sentence “In some embodiments, the composition comprises, consists essentially of, or consists of A” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition consists essentially of A. In some embodiments, the composition consists of A.”
  • a sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself.
  • the sentence “In some embodiments, the composition comprises A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C ” As another example, the sentence “In some embodiments, the composition comprises at least A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises at least A. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C.”
  • protein protein
  • polypeptide and “peptide” are used interchangeably to refer to two or more amino acids linked together. Groups or strings of amino acid abbreviations are used to represent peptides. Except when specifically indicated, peptides are indicated with the N-terminus on the left and the sequence is written from the N-terminus to the C-terminus. Except when specifically indicated, peptides are indicated with the N-terminus on the left and the sequences are written from the N-terminus to the C-terminus. Similarly, except when specifically indicated, nucleic acid sequences are indicated with the 5’ end on the left and the sequences are written from 5’ to 3’.
  • sequence identity refers to the percentage of nucleotides or amino acid residues that are the same between sequences, when compared and optimally aligned for maximum correspondence over a given comparison window, as measured by visual inspection or by a sequence comparison algorithm in the art, such as the BLAST algorithm, which is described in Altschul et al ., (1990) J Mol Biol 215:403-410.
  • Software for performing BLAST e.g ., BLASTP and BLASTN
  • analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov).
  • the comparison window can exist over a given portion, e.g., a functional domain, or an arbitrarily selection a given number of contiguous nucleotides or amino acid residues of one or both sequences.
  • the comparison window can exist over the full length of the sequences being compared. For purposes herein, where a given comparison window (e.g, over 80% of the given sequence) is not provided, the recited sequence identity is over 100% of the given sequence.
  • the percentages of sequence identity of the proteins provided herein are determined using BLASTP 2.8.0+, scoring matrix BLOSUM62, and the default parameters available at blast.ncbi.nlm.nih.gov/Blast.cgi. See also Altschul, et al, (1997) Nucleic Acids Res 25:3389-3402; and Altschul, etal, (2005) FEBS J 272:5101- 5109.
  • Optimal alignment of sequences for comparison can be conducted, e.g, by the local homology algorithm of Smith & Waterman, Adv Appl Math 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol Biol 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection.

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Abstract

L'invention concerne des méthodes pour augmenter ou diminuer les quantités d'interféron endogène chez des sujets, qui comprennent l'inhibition, la réduction, l'augmentation, l'amélioration ou la stabilisation de MORC3 chez les sujets.
PCT/US2022/016162 2021-02-12 2022-02-11 Méthodes pour induire une réponse à l'interféron par régulation de morc3 Ceased WO2022174065A1 (fr)

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US18/263,760 US20240076666A1 (en) 2021-02-12 2022-02-11 Immunomodulators Targeting MORC3 for Interferon Induction

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CN115466802A (zh) * 2022-10-28 2022-12-13 北京大学 Tp53bp2在调控干扰素信号通路及抗病毒中的用途
CN115466802B (zh) * 2022-10-28 2023-12-12 北京大学 Tp53bp2在调控干扰素信号通路及抗病毒中的用途
CN117746975A (zh) * 2023-12-22 2024-03-22 徐州医科大学 一种基于trim35和sting的计算机辅助药物筛选方法、系统及设备

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